Saturday, 24 October 2020

Plasmid research


 

Plasmids—extrachromosomal genetic elements—play an important role for bacteria. And with the importance of plasmids for antibiotic resistance, understanding them, their persistence and their interaction of chromosomes will be crucial in the next few decades.

 

The recent bioRxiv preprint "Evolutionary Mechanisms That Determine Which Bacterial Genes Are Carried on Plasmids" by Sonja Lehtinen, Jana S. Huisman and Sebastian Bonhoeffer builds a model where a bacterium "best" keeps its DNA, in the chromosome or in plasmids.

The model is realized as a set of time-dependent coupled differential equations. The stability of the equilibrium points of this system was analyzed using Mathematica. With its state-of-the-art symbolic and numeric linear algebra framework and its convenient-to-use data visualization functions, Mathematica is a well-suited tool for such computations.

In a related paper "Factors Favouring the Evolution of Multidrug Resistance in Bacteria" by Eliott Jacopin, Sonja Lehtinen, Florence Débarre and François Blanquart. Their paper deals with describing the equilibrium bacteria strain compositions. Similar to the previous paper, they used a coupled system of differential equations, and they also carried out all computations in Mathematica.


Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Thursday, 22 October 2020

How Does the Gut Microbiome Impact Potential SARS-CoV-2 Vaccines?


 

The novel SARS-CoV-2 strain’s high virulence has spread fear across the globe in this growing pandemic. The global impact and necessity for treatment has researchers racing to develop vaccines to protect us from COVID-19, and has raised greater awareness in the importance of vaccines and vaccine efficacy research advancement for global public health against infectious disease. 

A post from ThermoFisher.

To develop effective vaccines for SARS-CoV-2, it is important to understand host response and key factors that impact human immunity, protecting us from our external environment. One significant feature believed to influence human immunity is our gut microbiome. With hundreds of thousands of diverse bacteria dwelling in our gut, the gut microbiome is thought to be our second genome, having a myriad of effects on gene regulation of human immunity, metabolism and the central nervous system. However, one challenge that researchers face is understanding how the gut microbiome’s variations in diversity, such as reduction in microbial flora, may potentially impact susceptibility to infection and efficacy of various immune interventions such as vaccines. One solution to accurately understand microbiome diversity is to use next-generation sequencing (NGS) and its high throughput to capture genomic information across the entire microbial population for research. Unlike culture-based techniques, the high sensitivity of NGS can capture information of rare and environmentally sensitive taxa, including microbes that may not be culturable. This level of sensitivity is necessary to understand the complex interactions in the microbial community, as well as host-microbe relationships that may impact immune response and potentially effect vaccine efficacy.

In a recent paper, immunology researchers used NGS to characterize how antibiotic-mediated gut dysbiosis would affect influenza vaccine response. When healthy individuals received antibiotic treatment prior to vaccination, decreased microbial flora was observed for up to 180 days, as well as upregulated inflammation and impaired production of antibodies against particular influenza strains. Strains that were more similar to vaccines or flu exposure of previous years made no significant difference between immune responses between healthy and antibiotic administered individuals. However, H1N1, a virus where individuals had no previous antibody production or memory, resulted in a dampened immune response among the antibiotic administered individuals linked to reductions in their gut microbiome and in turn secondary metabolites related to immune response.

While this study utilizes NGS to tap into one of imperative interdisciplinary challenges of immunological research today, much work is to be done to examine microbially mediated immunological response not only healthy individuals, but also the elderly, infants, and immunocompromised individuals, understanding gut microbial diversity is of utmost importance going into the future. As the impact by SARs-CoV-2 continues to affect the global populous exponentially during this unprecedented time with vaccines still underway, targeted NGS utilizing the Ion AmpliSeq Microbiome Health Research Kit is a simplified and pertinent tool to understand and characterize microbially mediated immune health. With a highly sensitive workflow that utilizes eight of the nine hypervariable 16S rRNA gene regions to accurately detect population variation and taxon-specific identification, the Ion AmpliqSeq Microbiome Health Research Kit is an efficient yet comprehensive research tool to understand gut microbial shifts that could impact human health and efficacy of immune-modulatory drugs during this crucial time.

For more information on the latest microbiome panels for human research, please go to www.thermofisher.com/ngsmicrobiome.

Read the paper Thomas Hagan, et al., (2019) Antibiotics-Driven Gut Microbiome Perturbation Alters Immunity to Vaccines in Humans.

 

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Wednesday, 21 October 2020

Fluorescence–How CF Dyes Are An Evolutionary Step in Fluorescent Probes


 

How exactly are CF® dyes the next evolutionary step in fluorescent probes? CF dyes are very important in biological research applications. CF dyes are a series of water-soluble fluorescent dyes on the visible light and near-infrared light spectrums. What makes them so important, is they are used for labeling things like proteins, antibodies, nucleic acids, and other biomolecules. 

A guest post by Eric Torres

What Are the Mechanisms Behind Fluorescence?

A fluorophore is defined as any molecule that emits light of a certain wavelength after being excited by light of a different wavelength. The principle behind this process can be illustrated by a Jablonski diagram which describes a molecule’s electronic state (Figure 1.).

 

 

As a fluorescent molecule absorbs energy from incoming light particles (aka photons), the energy state is excited from a stable low energy ground state (S0), to several unstable higher energy levels. Within nanoseconds, some energy is lost to non-radiative emission and the molecule adopts a lower energy semi-stable excited state (S1).

The molecule will then return to ground state by the emission of light at a lower energy than the absorbed light. This shift in wavelength between absorption and emission is known as a Stokes shift.



Figure 1. Jablonski Diagram of excitation and emission of a fluorescent dye.
 

How is Brightness Measured?

A molecule’s brightness is determined by two metrics, molar absorptivity (ε) and quantum yield (Ф). The molar absorptivity, also known as a molar extinction coefficient, is a measure of how much light a molecule absorbs at a given wavelength. Meanwhile, the quantum yield is a ratio between how much light is emitted versus absorbed. The brightness of a molecule can be determined by multiplying the fluorescence quantum yield by the molar absorptivity.

Brightness = Quantum Yield (Ф) X Molar Absorptivity (ε)

Despite the simplicity of this equation, the process is a bit more complex. A molecule’s fluorescence profile and quantum yield can depend heavily on the environment. For example, dye-protein conjugations can lead to non fluorescent aggregates known as H-aggregates that drastically reduce brightness. This effect increases as more dye molecules are conjugated per protein. Other environmental factors that can influence a molecule’s fluorescence profile are the type of solvent used, dye concentration, and pH of the medium.

Fluorescence for Biology, A Delicate Balance

Brightness is just one piece of a much larger puzzle. In addition to fluorescence intensity, chemists must consider photostability, chemical stability, sources of background fluorescence, and other factors that determine how the dye will perform within a biological context. This poses a serious challenge for chemists, where chemical modifications intended to improve one characteristic may be detrimental to others.

Sulfonation supplies negative charges to the molecule which improves solubility and reduces dye aggregation. However, the negative charges also increase non-specific binding and background fluorescence. Essentially, we’ve traded one desirable attribute for another less desirable one.

So, where do we go from here?

Scientists at Biotium have pushed the envelope of modern fluorescence by developing CF® dyes, a new family of dyes with superior brightness, photostability and signal-to-noise. But what makes the CF® dyes so unique? Let’s focus on two novel chemical modifications that set CF® dyes apart from the competition: PEGylation and the rhodamine-imidazole substituent.
PEGylation, the Solution to Sulfonation

In 2007, Fei Mao and fellow chemists at Biotium were looking at possible solutions to the sulfonation issue. Their work attracted them to polyethyleneglycol (PEG), a polyether compound commonly used in life science research because it is non-immunogenic, water-soluble, and biologically inert. They hypothesized that PEG modification would help shield the negatively charged sulfonate groups, reducing the background from non-specific binding.

They were right.

PEG modification significantly diminished background fluorescence and enhanced the signal-to-noise of sulfonated dyes. But beyond amending issues with sulfonation, Biotium scientists knew the bulky PEG polymers would also help reduce dye aggregation, leading to increased quantum yield and fluorescence. In addition, PEG modifications further improved solubility, leading to increased biocompatibility for in vivo imaging. Today, pegylation and sulfonation are standard features for several of our CF® dyes, offering the best advantages from both modifications without compromise.

 

Figure 2. Evolution of cyanine dyes.


A “Bigger” Shift to Red

In 2009 Biotium made another breakthrough, this time centering on the rhodamine family of dyes. Until then, rhodamine dyes were known for their photostability, insensitivity to pH, and long emission wavelengths relative to fluorescein. However, despite extensive modifications, rhodamine dyes were unable to reach the longer near-IR emission wavelengths (>600 nm) that were capable of cyanine-based dyes.

Unfortunately, cyanine dyes were also less photostable and less soluble, limiting their utility. To solve this problem, Biotium scientists looked at several modifications that would extend the rhodamine fluorescence profile to longer wavelengths. Ironically, they found the answer not through modification of the xanthene core responsible for the dye’s color, but with the benzene group typically used for protein conjugation reactions.

By substituting the benzene ring with an imidazole group, Fei and his colleagues observed a 30 nm to 60 nm shift toward longer emission wavelengths. This discovery unlocked the potential of rhodamine dyes, offering the same photostability and chemical stability of xanthene-based dyes at near-IR wavelengths. Moreover, this substitution also improved solubility which led to increased brightness and biocompatibility.

This innovation allowed the development of CF®680R, a rhodamine-imidazole dye with unrivaled brightness and photostability. These attributes make CF®680R the dye of choice for super-resolution microscopy techniques, such as STORM and 3D super-resolution imaging, where photostability is critical.

 

 

Figure 3. Imidazole substitution of rhodamine-based dyes.


Clearer Fluorescence with CF® Dyes

After this in-depth look at the chemistry behind our CF® dyes, you might be wondering what does “CF” stand for? CF® was initially an abbreviation for “Cyanine-based Fluorescent dyes”. These were the first patented CF® dyes based on cyanine dye structures. 10 years and more than two dozen dyes later, the CF® dye portfolio encompasses multiple dye core structures spanning the fluorescence spectrum from UV to near-IR. Today, we believe “CF” more aptly stands for Clear Fluor: dyes that produce superior signal-to-noise.

At the end of the day, Biotium’s products are used to produce images representative of complex biological systems. Proper interpretation of those images requires clarity and trust in the signal the dye produces. With the chemical advancements described here we believe our dyes earn that trust. Today, our fluorescent CF® dyes lead the industry, offering superior brightness, photostability, and biocompatibility from blue to near-IR.

You can learn more about Biotium’s CF® dyes and their other innovative fluorescent technologies on their website.

http://www.pharmamicroresources.com/

Tuesday, 20 October 2020

Tackling foodborne infections

 


University of Helsinki researchers have been investigating the possibility of utilising phages to eradicate foodborne pathogens and preventing food poisoning. The researchers have focused on the Yersinia enterocolitica bacterium, by far the most common cause for the food poisioning disease yersiniosis. The disease is usually transmitted through raw or undercooked pork. Another source of infection, although a much rarer one, is milk. Humans can also be infected by kitchenware used in handling contaminated food. Yersiniosis symptoms include fever, severe abdominal pain and diarrhoea, which may persist for up to three weeks. In some cases, yersiniosis may cause arthritis as a secondary disease, persisting potentially several weeks. Yersiniosis occurs all over the world.

 

The scientists have identified four bacteriophages that infect the Y. enterocolitica bacterium. The most effective of this quartet proved to be the fHe-Yen9-01 phage. It was selected for the next stage of the study where its efficacy in decontaminating food and kitchenware contaminated by bacteria was investigated. Everyday products available in grocery shops, such as raw and grilled pork, as well as milk, were inoculated with Y. enterocolitica. The contaminated food was then subjected to phage treatment, after which the number of both bacteria and phages was monitored for three days. It was found that phage treatment was effective in inhibiting bacterial growth in food, while the number of phages in the food grew, indicating that phages infect bacteria and grow in them also when refrigerated.

 

See: Jun, J. W. Park, S. C., Wicklund, A. and Skurnik, M. (2018) Bacteriophages reduce Yersinia enterocolitica contamination of food and kitchenware. International Journal of Food Microbiology, 2018; DOI: 10.1016/j.ijfoodmicro.2018.02.007

 

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Monday, 19 October 2020

Impact of novel coronavirus SARS-CoV-2 in cleanroom operations


The novel coronavirus SARS-CoV-2 is causing problems globally. This includes cleanroom users. The virus can be passed in the air and it survives on surfaces for prolonged periods of time. While existing protective measures should minimise air risks (such as HEPA filters, air change rates, wearing masks and gloves) the surface risks, due to prolonged survival times, require careful selection of appropriate agents (primarily either alcoholic products at 61 to 71% concentration or hydrogen peroxide at 0.5% or higher).

In relation to this topic, Tim Sandle has written an article:

Sandle, T. (2020) Impact of novel coronavirus  SARS-CoV-2 in cleanroom operations, Industrial Pharmacy, 66: 6-9

For details, please contact Tim Sandle



Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Thursday, 15 October 2020

Synthetic Biology and Bioengineering Reshaping the Way We Think About Life



Think about a future where synthetic fish roam in rivers and other waterways, searching for germs to kill; where environment-friendly fuels and plastics are produced from yeast vats; where electronic systems repair themselves just like living beings do; and where viruses and other microbes are programmed to kill cancer cells.

This is the kind of world synthetic biology aims to build, where possibilities are far greater than imagination. For synthetic biology researchers, life is a machine and it can be designed and manufactured (yes, you read that correctly!) to solve various health, environmental, and energy problems existing in this big beautiful planet, we call the Earth. In simpler term, synthetic biology is either the building of genetic circuits, cells, enzymes, and several other biological entities, or the redesign of already existing living organisms.

According to the National Human Genome Research Institute, “Synthetic biology is a field of science that involves redesigning organisms for useful purposes by engineering them to have new abilities”. In the healthcare industry, synthetic biology promises to find cures for a plethora of diseases and health problems and make treatments highly effective. Moreover, rapid advancements in this domain in recent times have made it possible for many scientists and medical researchers to apply synthetic biology-based methods for designing diagnostics, developing molecularly engineered tissues, and building new vaccines and drugs.

Apart from designing new cures and treatments, synthetic biology can also help the clean environment and create a greener and cleaner Earth. In addition to this, it can enhance the efficiency in chemical technology and biomanufacturing and help build enzymes that can bolster the production of biofuels. Due to these benefits, popularity of synthetic biology is rising at an explosive pace, across the world, which is, in turn, causing the boom of the global synthetic biology market.

Soaring Investments Pushing Up Research and Development (R&D) Activities

Due to excellent clinical and environmental applications of synthetic biology, many public and private organizations are making huge investments in R&D activities in this domain. Some of the prominent organizations that are funding synthetic biology research are National Science Foundation (NSF), Centre for Chemical and Synthetic Biology (CCSB), International Association Synthetic Biology (IASB), Synthetic Biology Engineering Research Center (SynBERC), and National Aeronautics and Space Administration (NASA). Besides making huge investments in R&D projects, these organizations are also implementing initiatives for promoting basic and applied research.

Pharmaceuticals and Diagnostics, the Major Application Areas

Out of several areas and niches benefitting from synthetic biology, medicine is the largest one, especially with the large-scale usage of various strategies and tools developed in this domain. With the production of desired proteins that can target foreign or diseased cells and the development of engineered cells that can integrate multiple inputs and discriminate between cell types or states, synthetic biology is revolutionizing the way diseases are managed and treated. 
 


The biological systems developed by synthetic biologists are used in several aspects of healthcare and medicine, ranging from developing advanced cell-based therapeutic procedures that can provide personalized treatment and biologics, which can replace chemically-synthesized medicines to drug discovery. In future years, several more synthetic biology-based therapies and solutions are predicted to move from laboratory testing or concepts to clinical trials and approval.

Major Industries Being Transformed by Synthetic Biology


1. Biopharma: The adoption of synthetic biology is allowing companies operating in this industry to develop pathways that make possible for microbes to produce drugs, such as Taxol, which is an anti-cancer medicine, and Artemenisin, which is an antimalarial drug. Apart from this, synthetic biology can also improve the in-vitro drug production process. For example, Codexis utilizes synthetic biology for developing highly efficient enzymes for the synthesis of small molecule medicines.


2. Carbon Recycling: Synthetic biology has the ability to clean up the environment by reducing the emission levels and carbon footprint. This is mainly done by developing eco-friendly methods that can be used by companies using petroleum-based products. Furthermore, several companies are producing bioplastics or biofuels. For example, Synthetic Genomics is using algae for producing biofuels that can recycle the excess amounts of CO2 present in the atmosphere.


3. Fashion: The fashion industry is one of the biggest producers of carbon emissions. As a result, many fashion brands and companies are increasingly focusing on greener alternatives to the conventionally used chemically treated fabrics and dyes. Colorifix, a synthetic biology dye producing company, was developing methods via which blue jeans can be dyed without generating hazardous waste.



4. Cosmetics: The increasing concerns being raised over the usage of various animal products, such as collagen, in cosmetic items, including anti-wrinkle creams, are boosting the requirement for vegan-friendly and environment-friendly alternatives. As a result, many cosmetics companies are using synthetic biology for developing alternatives to products using animal collagen. For example, Biossance has recently developed squalene, which is an animal-free cosmetic, with the help of synthetic biology.


North America: Home of Synthetic Biology!

Globally, the adoption of synthetic biology solutions was the highest in North America during the past few years and this trend is likely to continue in the future as well. The main factors fueling the expansion of the market in this region are the rising incidence of chronic diseases and surging geriatric population. Apart from these factors, rapid technological advancements and soaring investments being made in synthetic biology research are also boosting the industry growth. In the region, usage of synthetic biology has been historically very high in the U.S.

Hence, it can be safely said that due to the rising R&D activities and growing requirement for advanced medicinal drugs and treatment procedures, on account of the rising incidence of chronic diseases and surging population of elderly people, across the world, the synthetic biology domain will flourish in coming years.


Source: P&S Intelligence

Tuesday, 13 October 2020

New study on coronavirus and surface survival


A new study of interest has been published in the Virology Journal “The effect of temperature on persistence of SARS-CoV-2 on common surfaces.”

 

This Australian study has looked at the survival rates of SARS-CoV-2 on surfaces, using initial viral loads broadly equivalent to the highest titres excreted by infectious patients. This found that viable virus was isolated for up to 28 days at 20°C from common surfaces such as glass, stainless steel and both paper and polymer banknotes.

 

Conversely, infectious virus survived less than 24 hours at 40°C on some surfaces.

 

At 20°C, the times taken to achieve a 90% reduction and a 50% reduction of viable viral particles are:

 

 

Surface

Average time for a 90% reduction

Average time for a 50% reduction

Stainless steel

6.0 days

1.8 days

Polymer bank note

6.9 days

2.1 days

Paper

9.1 days

2.8 days

Glass

6.3 days

1.9 days

Cotton

5.6 days

1.7 days

Vinyl

6.3 days

1.9 days

 

The researchers point out that the primary spread of the virus remains via aerosols and respiratory droplets, however contaminated surfaces also function as an important contributor in transmission of the virus.

 

It remains undetermined the degree of surface contact and the amount of virus required for infection.

 

Article link: https://virologyj.biomedcentral.com/articles/10.1186/s12985-020-01418-7

 

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Monday, 12 October 2020

The Digital Present And The Organization Of Work: How COVID-19 Has Forced Pharma To Reorganize


This article looks at some of the ways through which pharmaceutical firms have adapted and will continue to adapt going forwards, considering digital technologies (such as those required for remote working) to the reorganization of the workforce. 
 
  • Protect employees 
  • Ensure products are safe 
  • Continue with as many operations as possible, in order to maintain profitability  
The 2020 COVID-19 pandemic changed the outlook on remote work for many pharmaceutical and healthcare organizations around the world. This chapter looks at the changes that have impacted on the workplace, including the shift towards remote working. With remote working comes new opportunities as well as security threats.
 
The article follows on from two other coronavirus related articles by the author for the IVT Network.

The reference is:


Sandle, T. (2020) The Digital Present And The Organization Of Work: How COVID-19 Has Forced Pharma To Reorganize, The Journal of Validation Technology, 26 (3): https://www.ivtnetwork.com/article/digital-present-and-organization-work-how-covid-19-has-forced-pharma-reorganize

For further details, please contact Tim Sandle.

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

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